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. 2015 Mar 11;10(3):e0119577.
doi: 10.1371/journal.pone.0119577. eCollection 2015.

Angiopoietin-1 requires oxidant signaling through p47phox to promote endothelial barrier defense

Chandra C Ghosh  1 , Aditi Mukherjee  1 , Sascha David  2 , Katelyn E Milam  1 , Jon T Hunter  1 , Samir M Parikh  1
Affiliations

Angiopoietin-1 requires oxidant signaling through p47phox to promote endothelial barrier defense

Chandra C Ghosh et al. PLoS One. .

Abstract

Background: Reactive oxygen species (ROS) are largely considered to be pathogenic to normal endothelial function in disease states such as sepsis. We hypothesized that Angiopoietin-1 (Angpt-1), an endogenous agonist of the endothelial-specific receptor, Tie-2, promotes barrier defense by activating NADPH oxidase (NOX) signaling.

Methods and findings: Using primary human microvascular endothelial cells (HMVECs), we found that Angpt-1 stimulation induces phosphorylation of p47phox and a brief oxidative burst that is lost when chemical inhibitors of NOX activity or siRNA against the NOX component p47phox were applied. As a result, there was attenuated ROS activity, disrupted junctional contacts, enhanced actin stress fiber accumulation, and induced gap formation between confluent HMVECs. All of these changes were associated with weakened barrier function. The ability of Angpt-1 to prevent identical changes induced by inflammatory permeability mediators, thrombin and lipopolysaccharides (LPS), was abrogated by p47phox knockdown. P47phox was required for Angpt-1 to activate Rac1 and inhibit mediator-induced activation of the small GTPase RhoA. Finally, Angpt-1 gene transfer prevented vascular leakage in wildtype mice exposed to systemically administered LPS, but not in p47phox knock out (p47-/-) littermates.

Conclusions: These results suggest an essential role for NOX signaling in Angpt-1-mediated endothelial barrier defense against mediators of systemic inflammation. More broadly, oxidants generated for signal transduction may have a barrier-promoting role in vascular endothelium.

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Conflict of interest statement

Competing Interests: SMP advises Vasomune and Eunoia Biotech and Beth Israel Deaconess Medical Center lists SMP as an inventor on disclosures and patents pertaining to angiopoietins. Samir M. Parikh is member of the Editorial Board of PLoS One. Samir M. Parikh declares that Beth Israel Deaconess Medical Center has listed him as an inventor on a patent pertaining to angiopoietins (US 8,685,393). Samir M. Parikh confirms that the competing interests do not alter the authors’ adherence to all PLOS One policies on sharing data and materials regarding this manuscript.

Figures

Fig 1
Fig 1. Angpt-1 induces a p47phox-dependent oxidative burst in endothelium.
(A) Titration and validation of p47phox siRNA delivery into HMVECs. (B) Example epifluorescence (excitation 495 nm: emission 520 nm) and corresponding bright field images of individual HMVECs loaded with CM-H2DCFDA, pre-treated with Apo (650 μM), control siRNA, or p47phox siRNA, and then treated with Angpt-1 (300 ng/ml). Images shown were taken 5 minutes after Angpt-1 addition. Scale bar 10 μm. (C) Planimetric quantification of CM-H2DCFDA fluorescence from above conditions (n = 3–5 experiments per condition). ***p<0.001 compared to p47phox siRNA+Angpt-1. (D-G) Cells transfected with Hyper-3 for 48 hours, underwent serum starvation for 2 hours, were treated with 10 μM H2O2 or Angpt-1 (300 ng/ml), and changes in fluorescence intensity were measured by live cell imaging microscopy. Representative images after 2 minutes of H2O2 (D-E) and 5 minutes with Angpt-1 (F-G) are shown. (H) Hyper-3 transfected HMVECs were, treated with chemicals (Apo and VAS 2870), siRNA p47phox or Tie2-Fc (500 ng/ml) before addition of Angpt-1 (300 ng/ml). Results were analyzed by one-way ANOVA followed by post-hoc corrections for multiple comparisons. ***p<0.001,***p<0.01 relative to Angpt-1 alone.
Fig 2
Fig 2. Angpt-1 requires NOX activity to inhibit thrombin-induced RhoA activation.
Confluent HMVECs were treated with vehicle (NT), Angpt-1 (300 ng/ml), Apo (650 μM), and/or thrombin (1 U/ml). Cells were lysed in the manufacturer-provided buffer 15 minutes after treatments for RhoA G-LISA, a pulldown method for RhoA-GTP detection. The results were then normalized to total RhoA, as determined by densitometry of Western blots (n = 4–6 experiments per condition). *p< 0.05.
Fig 3
Fig 3. p47phox enables Angpt-1 to counteract cellular structural rearrangements induced by thrombin.
(A-H) Confluent HMVECs were treated with control siRNA, thrombin (1 U/ml, A-D), or the combination of Angpt-1 (300 ng/ml) plus thrombin (E-H). After 15 minutes, cells were fixed, permeabilized, and stained for nuclei (blue, DAPI), VE-cadherin (green), or F-actin (red). White arrows indicate paracellular gaps. (I-P) Repeat of the above experiments, replacing control siRNA with p47phox siRNA. Representative of n = 3–4 experiments per condition. Scale bar 10 μm.
Fig 4
Fig 4. p47phox is required for Angpt-1-mediated barrier defense against thrombin.
(A) Transendothelial resistance assay (TER) of confluent HMVECs treated with thrombin (1 U/ml), with p47phox or control siRNA, and with and without Angpt-1 (300 ng/ml). n = 4 experiments per condition. To enable comparisons between conditions, the baseline absolute resistance of each well was used to normalize subsequent readings for the respective well. (B) Data from (A) quantified as the change in normalized resistance 30 minutes after thrombin addition. ***p<0.001. (C) Transendothelial resistance assay (TER) of control- vs. p47phox-siRNA-treated HMVECs to which LPS (10 ng/ml) and Angpt-1 (300 ng/ml) were applied. The change was recorded 1 hour after LPS and Angpt-1 addition (n = 3 experiments per condition). ***p< 0.001, n = 4 experiments per condition.
Fig 5
Fig 5. Angpt-1 mediated barrier defense in acute systemic inflammation requires p47phox.
(A) Spectrophotometric quantification at 620 nm of intravenously injected Evans blue dye extravasation into the lungs of wildtype littermates (p47+/+) and p47phox KO mice (p47−/−) 16 hours after LPS (15 mg/kg IP) with prior control adenovirus (control) or Angpt-1 adenovirus (1 x 109pfu/mouse) gene transfer. (B-D) Lung photomicrographs from above conditions (representative of n = 3–5 mice per condition). Scale bar 50 μm.
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